In general, the invention relates to in vitro techniques for detecting interactions of proteins with other proteins or compounds, and for selecting such interacting proteins.
In eukaryotic cells, the expression of individual genes is tightly regulated on the level of mRNA transcription. The cellular transcription machinery consists of a multi-component complex that includes RNA polymerase II and several associated proteins (Tjian & Maniatis, Cell (1994) vol. 77, p. 5-8). Transcription is initiated by binding of the polymerase complex to a core promoter sequence located upstream of the corresponding gene. This binding process is greatly facilitated by transcription activators that bind to DNA sites outside the core promoter and recruit the polymerase complex through additional non-covalent interactions (Ptashne & Gann, Nature (1997) vol. 386, p. 569-77).
In order to be effective, the linkage between the DNA-binding and activation domains of transcription activation factors need not be covalent; indeed, these domains function even when present on separate (but associated) molecular entities. Proximity between the DNA-binding and activator portion of a transcription factor can be restored by either direct dimerization of the components (“A” and “B” in FIG. 1) or through the action of adaptor or mediator molecules (“M” in FIG. 2). Successful gene expression is dependent on the corresponding association event and can serve as a means to test for molecular interactions between those domains. Even relatively weak molecular interactions and those with fast off-rates can trigger transcription over time and can therefore be detected.
Based on this, a number of “two-hybrid” (FIG. 1A; Fields & Song, Nature (1989) vol. 340, p. 245-246) and “three-hybrid” systems (FIG. 2A; Sengupta et al., Proc. Natl. Acad. Sci. USA (1996) vol. 93, p. 8496-8501; Lictria & Liu, Proc. Natl. Acad. Sci. USA (1996) vol. 93, p. 12817) have been devised for the identification of protein/protein and drug/protein interactions (for reviews see e.g.: Kolanus, Curr Top Microbiol Immunol. (1999) vol. 243, p. 37-54; Vidal & Legrain, Nucleic Acids Res. (1999), vol. 27, p. 4; Drees, Curr. Opinion Chem. Biol. (1999) vol. 3, p. 64-70). These assays, however, are somewhat restricted when applied to the screening of peptide or protein libraries. Since genotype (i.e, the plasmid encoding an individual library member) and phenotype (i.e., the protein library member itself) are not covalently linked, their association must be maintained within the confines of a cell. Accordingly, these assays must be performed in in vivo expression systems, generally, in yeast cells. This imposes several limitations resulting, for example, from the restriction of effective library size by transformation efficiencies, the occurrence of unwanted selection pressure inside a cellular environment, the need to exclude molecules that exhibit toxic effects on cells, and the difficulty in delivering certain drugs to the cytoplasm of cells (see, for example, Roberts, Curr. Opin. Biotech. (1999) vol. 3, p. 268-273). Accordingly, new approaches to the detection of protein/protein and protein/compound interactions are desirable.